1. Field of the Invention
The present invention relates to a method for producing L-cysteine or a related substance thereof. L-cysteine and related substances thereof are used in the fields of drugs, cosmetics, and foods.
2. Brief Description of the Related Art
L-cysteine is conventionally obtained by extraction from keratin-containing substances such as hair, horns, and feathers, or converting the precursor DL-2-aminothiazoline-4-carboxylic acid using a microbial enzyme. L-cysteine can also be produced on a large scale by an immobilized enzyme method utilizing a novel enzyme. Furthermore, L-cysteine can also be produced by fermentation utilizing a microorganism.
The ability of a microorganism to produce L-cysteine can be improved by enhancing the activity of an enzyme of the L-cysteine biosynthesis pathway, or an activity of an enzyme involved in generation of a compound which acts as a substrate of that pathway, such as L-serine. Microorganisms which are able to produce L-cysteine include, for example, a coryneform bacterium in which intracellular serine acetyltransferase activity is increased (Japanese Patent Laid-open (Kokai) No. 2002-233384). L-cysteine-producing ability can also be increased by incorporating a serine acetyltransferase which has been mutated so that feedback inhibition by L-cysteine is attenuated (Japanese Patent Laid-open (Kokai) No. 11-155571, U.S. Patent Published Application No. 20050112731 and U.S. Pat. No. 6,218,168). A mutant serA gene coding for a mutant 3-phosphoglycerate dehydrogenase of which feedback inhibition by serine is attenuated is also known, and use thereof in production of L-cysteine by Escherichia coli has been suggested (U.S. Pat. No. 5,856,148 and U.S. Patent Published Application No. 20050009162).
Furthermore, L-cysteine-producing ability of a microorganism can also be improved by suppressing the system which acts to decompose L-cysteine. Microorganisms in which this system has been suppressed, and the L-cysteine-producing ability is enhanced, include coryneform bacteria or Escherichia bacteria in which activity of cystathionine-β-lyase (Japanese Patent Laid-open (Kokai) No. 11-155571), tryptophanase (Japanese Patent Laid-open (Kokai) No. 2003-169668), or O-acetylserine sulfhydrylase B (Japanese Patent Laid-open (Kokai) No. 2005-245311) is attenuated or deleted.
Moreover, L-cysteine-producing ability of a microorganism can also be improved by enhancing the L-cysteine-secreting ability. For example, techniques have been reported of enhancing L-cysteine-producing ability by enhancing expression of the ydeD gene (Dassler et al., Mol. Microbiol., 36, 1101-1112 (2000)), yfiK gene (Macdonald and Cole, Molecular and General Genetics, 200(2):328-334 (1985)), or yeaS gene (Warren et al., Biochemical Journal, 265(3):725-729 (1990)), which codes for a protein that participates in secretion of L-cysteine. Furthermore, techniques have been reported of enhancing L-cysteine-producing ability by enhancing expression of the mar locus, emr locus, acr locus, cmr locus, mex gene, bmr gene or qacA gene (U.S. Pat. No. 5,972,663), or emrAB, emrKY, yojIH, acrEF, bcr or cusA gene (Japanese Patent Laid-open (Kokai) No. 2005-287333). These loci/genes encode proteins which are responsible for secreting a substance which is cytotoxic from the cells.
L-cysteine is a sulfur-containing amino acid, and therefore, metabolism of a sulfur source is involved in the production of L-cysteine. The biosynthesis pathway of L-cysteine when glucose is the carbon source and the sulfate or thiosulfate ion is the sulfur source, and examples of genes involved in that pathway are shown in FIG. 1.
Sulfite reductase is an enzyme that catalyzes, in the sulfate reduction pathway (Frederich C., Neidhardt et al., Escherichia Coli and Salmonella Cellular and Molecular Biology, 2nd Edition, Vol. 1, ASM Press, 514-527) in which sulfate ion (SO42−) is converted into sulfide ion (—S2−), the reaction of reducing sulfite ion (—SO32−) into sulfide ion (—S2−) as the final step. The sulfide ion generated from the sulfate ion via the sulfate reduction pathway reacts with O-acetylserine (OAS) to generate L-cysteine. That is, when L-cysteine is produced with sulfate ions as the sulfur source, the sulfite reductase is considered to be one of the enzymes that participate in the L-cysteine biosynthesis. It is known that the sulfite reductase of Escherichia coli has an α8β4 complex structure which includes a subunits encoded by the cyst gene and β subunits encoded by the cysI gene. Furthermore, the cysG gene codes for the biosynthesis enzyme of siroheme, which is a cofactor of the sulfite reductase β subunit. The cysJ gene and the cysI gene are present on the same operon, whereas the cysG gene is on a different site of the genome. The details of the cysJ gene, the cysI gene and the proteins encoded by these genes are described in Ostrowski et al., Journal of Biological Chemistry, 264(27):15796-15808 (1989), Li et al., Gene, 53(2-3):227-234 (1987), Gaudu and Fontecave, European Journal of Biochemistry, 226 (2):459-463 (1994), Eschenbrenner et al., Journal of Biological Chemistry, 270(35):20550-20555 (1995) and Ostrowski et al., Journal of Biological Chemistry, 264(26):15726-15737 (1989). Furthermore, the details of the cysG gene and the protein encoded by this gene are described in Peakman et al., European Journal of Biochemistry, 191(2):315-323 (1990), Macdonald and Cole, Molecular and General Genetics, 200(2):328-334 (1985), Warren et al., Biochemical Journal, 265(3):725-729 (1990) and Spencer et al., FEBS Letters 335(1):57-60 (1993).
Although there has been no report directly linking enhancing expression of the cysG gene, cysJ gene or cysI gene with increased L-cysteine production, an Escherichia coli bacteria in which activity of a protein encoded by the cysB gene is enhanced is known as an L-cysteine-producing bacterium (International Patent Publication WO01/27307). Because the protein encoded by the cysB gene positively regulates expression of the cysJIH operon which includes the cysJ gene and cysI gene coding for sulfite reductase, expression of the cysJ gene and cysI gene may be increased in that Escherichia coli. On the other hand, expression of the cysG gene is not regulated by the protein encoded by the cysB gene, and enhancement of expression of the cysG gene for L-cysteine production has not been reported.
Furthermore, the effectiveness of enhancing expression of the cysG gene, cysJ gene or cysI gene in the production of amino acids other than L-cysteine has been suggested. For example, enhancing expression of the cys genes including the cysG gene, cysJ gene and cysI gene, is referred to concerning production of L-threonine or L-lysine (U.S. Pat. No. 7,759,094), production of L-threonine (International Patent Publication WO03/006666), and production of L-methionine (U.S. Patent Published Application No. 2009298136 and International Patent Publication WO2008/127240). Furthermore, enhancing expression of the cys genes including the cysJ gene and the cysI gene is also referred to concerning production of L-methionine (International Patent Publication WO2009/043372, International Patent Publication WO2005/108561, International Patent Publication WO2007/077041, and International Patent Publication WO2006/082254). Moreover, enhancing expression of the cysJIH operon including the cysJ gene and the cysI gene is also referred to concerning production of L-methionine (U.S. Patent Published Application No. 20100047879). Furthermore, increasing L-arginine production by enhancing expression of the cysG gene has been reported (U.S. Patent Published Application No. 20050069994).
On the other hand, thiosulfate is metabolized in a pathway other than the sulfate reduction pathway (FIG. 1), and it is not known whether sulfite reductase participates in the metabolism of thiosulfate. Furthermore, all the aforementioned findings concerning production of amino acids fail to suggest that enhancing expression of the cysG gene, cysJ gene or cysI gene can be effective for amino acid production using thiosulfate as the sulfur source. As described above, the relationship between production of an amino acid such as L-cysteine using thiosulfate as the sulfur source and the cysG gene, cysJ gene or cysI gene has not been previously reported.